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Denitrifying woodchip bioreactors (WBRs) are increasingly used to manage the release of non‐point source nitrogen (N) by stimulating microbial denitrification. Woodchips serve as a renewable organic carbon (C) source, yet the recalcitrance of organic C in lignocellulosic biomass causes many WBRs to be C‐limited. Prior studies have observed that oxic–anoxic cycling increased the mobilization of organic C, increased nitrate (NO3−) removal rates, and attenuated production of nitrous oxide (N2O). Here, we use multi‐omics approaches and amplicon sequencing of fungal 5.8S‐ITS2 and prokaryotic 16S rRNA genes to elucidate the microbial drivers for enhanced NO3− removal and attenuated N2O production under redox‐dynamic conditions. Transient oxic periods stimulated the expression of fungal ligninolytic enzymes, increasing the bioavailability of woodchip‐derived C and stimulating the expression of denitrification genes. Nitrous oxide reductase (nosZ) genes were primarily clade II, and the ratio of clade II/clade I nosZ transcripts during the oxic–anoxic transition was strongly correlated with the N2O yield. Analysis of metagenome‐assembled genomes revealed that many of the denitrifying microorganisms also have a genotypic ability to degrade complex polysaccharides like cellulose and hemicellulose, highlighting the adaptation of the WBR microbiome to the ecophysiological niche of the woodchip matrix. [ABSTRACT FROM AUTHOR]

Summary: Bradyrhizobia are common members of soil microbiomes and known as N2‐fixing symbionts of economically important legumes. Many are also denitrifiers, which can act as sinks or sources for N2O. Inoculation with compatible rhizobia is often needed for optimal N2‐fixation, but the choice of inoculant may have consequences for N2O emission. Here, we determined the phylogeny and denitrification capacity of Bradyrhizobium strains, most of them isolated from peanut‐nodules. Analyses of genomes and denitrification end‐points showed that all were denitrifiers, but only ~1/3 could reduce N2O. The N2O‐reducing isolates had strong preference for N2O‐ over NO3−‐reduction. Such preference was also observed in a study of other bradyrhizobia and tentatively ascribed to competition between the electron pathways to Nap (periplasmic NO3− reductase) and Nos (N2O reductase). Another possible explanation is lower abundance of Nap than Nos. Here, proteomics revealed that Nap was instead more abundant than Nos, supporting the hypothesis that the electron pathway to Nos outcompetes that to Nap. In contrast, Paracoccus denitrificans, which has membrane‐bond NO3− reductase (Nar), reduced N2O and NO3− simultaneously. We propose that the control at the metabolic level, favouring N2O reduction over NO3− reduction, applies also to other denitrifiers carrying Nos and Nap but lacking Nar. [ABSTRACT FROM AUTHOR]

Summary: This study coupled a landscape‐scale metagenomic survey of denitrification gene abundance in soils with in situ denitrification measurements to show how environmental factors shape distinct denitrification communities that exhibit varying denitrification activity. Across a hydrologic gradient, the distribution of total denitrification genes (nap/nar + nirK/nirS + cNor/qNor + nosZ) inferred from metagenomic read abundance exhibited no consistent patterns. However, when genes were considered independently, nirS, cNor and nosZ read abundance was positively associated with areas of higher soil moisture, higher nitrate and higher annual denitrification rates, whereas nirK and qNor read abundance was negatively associated with these factors. These results suggest that environmental conditions, in particular soil moisture and nitrate, select for distinct denitrification communities that are characterized by differential abundance of genes encoding apparently functionally redundant proteins. In contrast, taxonomic analysis did not identify notable variability in denitrifying community composition across sites. While the capacity to denitrify was ubiquitous across sites, denitrification genes with higher energetic costs, such as nirS and cNor, appear to confer a selective advantage in microbial communities experiencing more frequent soil saturation and greater nitrate inputs. This study suggests metagenomics can help identify denitrification hotspots that could be protected or enhanced to treat non‐point source nitrogen pollution. [ABSTRACT FROM AUTHOR]

Model denitrifiers convert NO-3 to N2, but it appears that a significant fraction of natural populations are truncated, conducting only one or two steps of the pathway. To better understand the diversity of partial denitrifiers in soil and whether discrepancies arise between the presence of known N-oxide reductase genes and phenotypic features, bacteria able to reduce NO-3 to NO-2 were isolated from soil, N-oxide gas products were measured for eight isolates, and six were genome sequenced. Gas phase analyses revealed that two were complete denitrifiers, which genome sequencing corroborated. The remaining six accumulated NO and N2O to varying degrees and genome sequencing of four indicated that two isolates held genes encoding nitrate reductase as the only dissimilatory N-oxide reductase, one contained genes for both nitrate and nitric oxide reductase, and one had nitrate and nitrite reductase. The results demonstrated that N-oxide production was not always predicted by the genetic potential and suggested that partial denitrifiers could be readily isolated among soil bacteria. This supported the hypothesis that each N-oxide reductase could provide a selectable benefit on its own, and therefore, reduction of nitrate to dinitrogen may not be obligatorily linked to complete denitrifiers but instead a consequence of a functionally diverse community. [ABSTRACT FROM AUTHOR]

The ctaD gene encoding subunit I of the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides has been cloned. The gene encodes a polypeptide of 565 residues which is highly homologous to the sequences of subunit I from other prokaryotic and eukaryotic sources, e.g. 51% identity with that from bovine, and 75% identity with that from Paracoccus denitrificans. The ctaD gene was deleted from the chromosome of R/ sphaeroides, resulting in a strain that spectroscopically lacks cytochrome a. This strain maintains about 50% of the cytochrome c oxidase activity of the wild-type strain owing to the presence of an alternate o-type cytochrome c oxidase. The aa3-type oxidase was restored by complementing the chromosomal deletion with a plasmid-borne copy of the ctaD gene. This system is well suited for site-directed mutagenesis probing of the structure and function of cytochrome c oxidase. [ABSTRACT FROM AUTHOR]